The altered metabolism of vitamin D3 during pregnancy will be studied with particular reference to the formation of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in placental tissue. Formation of 1,25(OH)2D3 has been observed when cells isolated from D-depleted rats on the twenty first day of gestation were incubated in a serum-free system with tritiated 25-hydroxyvitamin D3 (25OHD3). Selected aspects of this rat placental 25OHD3-1Alpha-hydroxylase will be determined, such as subcellular localization, cofactor requirements and kinetic parameters, for comparison with values obtained from similar studies of the renal hydroxylase. These experiments will be carried out by in vitro incubations of subcellular fractions and isolated cells using 3H-25OHD3 altering either cofactor or substrate concentrations accordingly. Once the enzyme has been described further studies will focus on in vivo and in vitro regulation of enzyme activity. Cells will be isolated from placenta of rats at earlier stages of gestation and also from placenta of rats subjected to various dietary treatments such as calcium or phosphorus deprivation. Enzyme activity in these cells will be measured by incubation in a serum-free system with 3H-25OHD3 and compared to that in cells from control rats. To facilitate the evaluation of direct effects of hormones and ions on the placental hydroxylase a system will be developed to maintain viable rat placental cells for 3 to 5 days. Initial efforts will be directed toward plating of cells for primary culture according to techniques used with human placental cells. Hormones will be added directly to culture plates according to appropriate time courses. Serum-containing media, if used in culture process, will be replaced by serum-free media at least 8 hours prior to incubation of cells with 3H-25OHD3. Such a system would allow extensive studies of in vitro effects of hormones such as parathyroid hormone, placental lactogen and prolactin as well as long term alterations of ionic content of media. D-metabolites will be extracted from the incubated media and cells and analyzed both quantitatively and qualitatively by high performance liquid chromatography and radioactive counting. The long term objectives of these studies are to determine the function of this extrarenal 25OHD3-1Alpha-hydroxylase, the factors that regulate hydroxylase activity and how increased 1,25(OH)2D3 may relate to normal fetal development and mineral homeostasis.